Blood coagulation time measurement method

Abstract

Provided is a method for precisely measuring the coagulation time of a blood sample exhibiting various coagulation reactions. This blood coagulation time measurement method includes measuring a coagulation reaction regarding a sample obtained by mixing a test sample and a coagulation time measurement reagent, calculating a weighted average time for a calculation target region of a waveform relating to coagulation speed from the obtained measurement data, and determining the weighted average time as the blood coagulation time.

Description

[Technical field]

[0001] The present invention relates to a method for measuring blood coagulation time. [Background technology]

[0002] A blood coagulation test is a test to diagnose a patient's blood coagulation ability by adding a specific reagent to a blood sample from the patient and measuring the blood coagulation time. Typical examples of blood coagulation times include the prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time. A blood coagulation test can examine a patient's hemostatic and fibrinolytic abilities. Abnormalities in blood coagulation ability mainly cause an increase in clotting time. For example, an increase in clotting time can be caused by the effects of coagulation inhibitors, a decrease in components involved in coagulation, a congenital deficiency of blood coagulation factors, or an acquired autoantibody that inhibits the coagulation reaction.

[0003] In recent years, automatic analyzers that perform automatic measurements of blood coagulation tests have been widely used, making it possible to easily perform blood coagulation tests. For example, in some types of automatic analyzers, a light is applied to a mixed solution obtained by adding a reagent to a blood sample, and the coagulation reaction of the blood sample is measured based on the change in the amount of light obtained. For example, when the amount of scattered light is measured, the amount of scattered light increases rapidly due to the progress of coagulation after a certain amount of time has passed since the addition of the reagent to the blood sample, and then, as the coagulation reaction approaches completion, the amount of scattered light saturates and reaches a plateau. The blood coagulation time can be measured based on such a change in the amount of scattered light over time.

[0004] Several methods, such as the percentage method and the differential method, are used to calculate the clotting time using an automatic analyzer (see Patent Document 1). When calculating the clotting time based on the amount of scattered light, the percentage method typically calculates the clotting time as the time until the measured amount of scattered light reaches a certain percentage of its maximum value. The percentage method allows for fairly accurate calculation of the clotting time not only for normal samples, but also for abnormal samples such as low fibrinogen samples, chyle samples, and hemolytic samples. On the other hand, automatic analysis based on the percentage method requires a long sample measurement time to be set so that the maximum amount of scattered light can be detected even in abnormal samples with low clotting ability, such as low fibrinogen samples, and therefore takes a long time to analyze.

[0005] In the differential method, the clotting time is typically calculated as the time until the differential value of the scattered light reaches a peak or a certain percentage of the peak. However, in abnormal samples with low clotting ability, such as low fibrinogen samples, the differential value of the scattered light may not show a clear peak. Furthermore, in abnormal samples, the differential value may show two or more peaks. A method is sometimes used to calculate the clotting time based on a single-peak curve created by fitting the differential curve, but fitting may lose accurate information about the clotting ability of the sample.

[0006] Furthermore, photometric data from an analyzer contains various noises due to the condition of the device, reagents, and sample, which may result in erroneous detection of the clotting time. In the automated analysis of blood samples, it is necessary to calculate a reliable clotting time by removing the adverse effects of noise. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 6-249855 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to a method for measuring blood coagulation time, which can accurately measure the coagulation time of a blood specimen showing various blood coagulation reaction curves.

Means for Solving the Problems

[0009] That is, the present invention provides the following. 〔1〕A method for measuring blood coagulation time, comprising: measuring a coagulation reaction of a sample obtained by mixing a test specimen and a coagulation time measurement reagent; calculating a weighted average time for an arithmetic operation target region of a waveform related to a coagulation rate from the obtained measurement data; and determining the blood coagulation time of the test specimen based on the weighted average time, wherein the arithmetic operation target region is a region where the waveform related to the coagulation rate is equal to or greater than a predetermined lower limit value in the waveform related to the coagulation rate. A method. 〔2〕The method according to 〔1〕, wherein the waveform related to the coagulation rate is a coagulation reaction curve or a first derivative curve of a relative value thereof. 〔3〕When the weighted average time is the time t1 and t2 (t1 < t2) when the waveform related to the coagulation rate is F(t) (t is time) and F(t) is x% of the maximum value (x is a predetermined value set in the range of 5 to 95), the following formula:

Number

[0010] According to the method of the present invention, it is possible to accurately measure the clotting time of blood samples that show various blood clotting reaction curves, including normal and abnormal samples. Furthermore, when analyzing a large number of samples in real time using an automatic analyzer, the method of the present invention can shorten the analysis time per sample and improve the analysis efficiency compared to the conventional percentage method. [Brief description of the drawings]

[0011] [Figure 1] 2 shows a basic flow chart of one embodiment of the blood coagulation time measuring method according to the present invention. [Diagram 2] 2 shows an embodiment of the procedure for the data analysis process shown in FIG. 1. [Diagram 3] An example of a clotting reaction curve. [Figure 4] An example of a coagulation reaction curve after pretreatment. [Diagram 5] A: A partially enlarged view of an example of a coagulation reaction curve. B: A partially enlarged view of an example of a coagulation reaction curve after pretreatment. [Figure 6] An example of a corrected zero order curve. [Figure 7] An example of a corrected linear curve. [Figure 8] FIG. 1 is a conceptual diagram showing the calculation area and weighted average score. [Figure 9] A conceptual diagram showing the change in weighted average score depending on the area to be calculated. [Figure 10] Linear regression line of vT20% at 20% calculation range against APTT by percentage method. [Figure 11] Slope (A), intercept (B), and correlation coefficient (C) of the linear regression line of vT in the 5-95% calculation range (vT5%-vT95%) against APTT by the percentage method. [Figure 12]Errors of weighted mean times (vT5%-vT95%) in the 5-95% calculation range for 24 samples relative to the control (APTT by percentage method). The shaded cells in the table indicate that the difference between the weighted mean time and the control is within ±5% (A) and ±2.5% (B) of the control. [Figure 13] Linear regression line of vT30% in the 30% calculation range against PT using the percentage method. [Figure 14] Slope (A), intercept (B), and correlation coefficient (C) of the linear regression line of vT in the 5 to 95% calculation range (vT5% to vT95%) against PT by the percentage method. [Figure 15] Errors of weighted mean times (vT5%-vT95%) in the 5-95% calculation range for 23 samples relative to the control (PT by percentage method). The shaded cells in the table indicate that the difference between the weighted mean time and the control is within ±5% (A) and ±2.5% (B) of the control. [Figure 16] Linear regression line of [Fbg] calculated using the weighted average time in the 35% calculation range against [Fbg] calculated using the percentage method. [Figure 17] Slope (A), intercept (B), and correlation coefficient (C) of the linear regression line of [Fbg] calculated based on vT in the 5 to 95% calculation range (vT5% to vT95%) to [Fbg] calculated by the percentage method. [Figure 18] Errors of calculated [Fbg] values ​​in the 5-95% calculation range for concentration series sample data of 20 samples, relative to the expected value. The grey cells in the table indicate that the error between the calculated value and the expected value is within ±10% (A) and ±5% (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In a blood coagulation test, a specific reagent is added to a blood sample, the blood coagulation reaction is measured, and the blood coagulation time is measured from the coagulation reaction. In the following specification, the blood sample may be referred to as a sample. A general means is used to measure a blood coagulation reaction, for example, an optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or a mechanical means for measuring the viscosity of plasma. The coagulation reaction curve of a normal sample basically shows a sigmoid shape, depending on the measurement means. For example, the coagulation reaction curve based on the amount of scattered light of a normal sample usually rises sharply due to the progress of coagulation after a certain amount of time has passed since the addition of the reagent, and then reaches a plateau as the coagulation reaction approaches completion. On the other hand, the coagulation reaction curve of an abnormal sample having a coagulation abnormality shows various shapes depending on the cause of the abnormality, such as a delay in the rise time of the curve or a gradual rise. The variety of coagulation reaction curves of abnormal samples makes it difficult to accurately measure the coagulation time with an automatic analyzer.

[0013] In a conventional general blood clotting time measurement, data is acquired at least until the end of the clotting reaction, and the clotting time is calculated based on the acquired data. For example, in the case of calculating the clotting time based on the amount of scattered light, the end of the clotting reaction is judged to be when the amount of scattered light is saturated, and then the clotting time is determined to be when the clotting reaction curve reaches the maximum speed or 1 / N of the maximum speed between the time of adding the reagent and the time of the end of the clotting reaction (differential method), and the clotting time is determined to be when the amount of scattered light reaches 1 / N of the time of the end of the clotting reaction (percentage method, see Patent Document 1). However, due to the abnormal shape or noise of the clotting reaction curve of the abnormal sample as described above, erroneous detection of the peak of the clotting reaction rate or the end of the clotting reaction may occur, and for example, the peak of the reaction rate or the end of the reaction may be detected too early. Such erroneous detection leads to an inaccurate calculation of the clotting time.

[0014] In an automatic analyzer, in order to efficiently analyze a large number of samples, it is desirable to end the measurement of one sample as soon as necessary data is obtained and start the measurement of the next sample. However, such a method has the risk of prematurely ending the measurement due to the above-mentioned false detection of the end of the clotting reaction, resulting in missing necessary data. On the other hand, if the clotting reaction measurement time per sample is fixed to a sufficiently long time, it is possible to prevent missing data due to false detection of the end of the clotting reaction. However, such a method reduces the overall analytical efficiency because the measurement time is longer than necessary for many samples.

[0015] The present invention prevents erroneous detection of clotting time due to the abnormal shape of the clotting reaction curve as described above, and enables accurate measurement of clotting time. Furthermore, according to the present invention, the minimum clotting reaction measurement time required for each clotting time measurement can be applied to various blood samples, including normal samples and abnormal samples, thereby shortening the analysis time per sample.

[0016] [Blood coagulation time measurement method] The present invention relates to a method for measuring the blood clotting time of a blood sample. The blood clotting time measuring method of the present invention (hereinafter also referred to as the method of the present invention) includes measuring the clotting reaction of a sample obtained by mixing a test sample with a clotting time measuring reagent, and calculating a weighted average time for a predetermined calculation range of a waveform related to the clotting rate from the obtained measurement data. One embodiment of the method of the present invention will be described with reference to FIG. 1. In this method, a sample is first prepared from the test sample, and then a clotting reaction measurement is performed for the sample (step 1). The obtained measurement data is analyzed to obtain a waveform related to the clotting rate of the sample, and then a weighted average time for a predetermined calculation range of the waveform is calculated (step 2). The clotting time of the test sample is determined based on the obtained weighted average time (step 3).

[0017] 1. Clotting reaction measurement In the coagulation reaction measurement, the coagulation reaction of a test sample mixed with a reagent is measured. From the time series data of the coagulation reaction obtained by this measurement, the blood coagulation time is measured. Examples of blood coagulation times measured by the method of the present invention include prothrombin time (PT), activated partial thromboplastin time (APTT), and coagulation time in fibrinogen concentration (Fbg) measurement. In the following specification, the method of the present invention will be mainly explained by taking activated partial thromboplastin time (APTT) as an example of coagulation time. Those skilled in the art can modify the method of the present invention to other coagulation times (e.g., prothrombin time (PT)).

[0018] In the method of the present invention, the subject's plasma is preferably used as the test blood sample. An anticoagulant commonly used in coagulation tests may be added to the sample. For example, blood is collected using a blood collection tube containing sodium citrate, and then centrifuged to obtain plasma.

[0019] A coagulation time measuring reagent is added to the test specimen to initiate a blood coagulation reaction. The coagulation reaction of the mixture after the addition of the reagent can be measured. The coagulation time measuring reagent used can be selected arbitrarily according to the purpose of measurement. Various reagents for measuring coagulation times are commercially available (for example, APTT reagent Coagpia APTT-N; manufactured by Sekisui Medical Co., Ltd.). A general means, for example, an optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or a mechanical means for measuring the viscosity of plasma, may be used to measure the coagulation reaction. The start time of the coagulation reaction can typically be defined as the time when the coagulation reaction is initiated by mixing the reagent with the specimen, but other timings may also be defined as the start time of the reaction. The time for which the measurement of the coagulation reaction is continued can be, for example, several tens of seconds to about 7 minutes from the time when the specimen and the reagent are mixed. This measurement time may be a fixed value determined arbitrarily, or may be until the end of the coagulation reaction of each specimen is detected. During the measurement time, the progress of the coagulation reaction can be repeatedly measured (photometrically if optically detected) at a predetermined interval. For example, the measurement may be performed at 0.1 second intervals. The temperature of the mixture during the measurement is under normal conditions, for example, 30°C or higher and 40°C or lower, preferably 35°C or higher and 39°C or lower. In addition, various measurement conditions can be appropriately set depending on the test specimen, reagent, measurement means, etc.

[0020] A series of operations in the above-mentioned coagulation reaction measurement can be performed using an automatic analyzer. One example of an automatic analyzer is the blood coagulation automatic analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations may be performed manually. For example, the preparation of the test sample may be performed manually, and the subsequent operations may be performed by the automatic analyzer.

[0021] 2. Data Analysis 2.1) Data pre-processing and correction Next, the data analysis in step 2 will be described. The flow of data analysis is shown in Fig. 2. The data analysis in step 2 may be performed in parallel with the coagulation reaction measurement in step 1, or may be performed later using data from a coagulation reaction measurement obtained in advance. Preferably, the data analysis in step 2 is performed in parallel with the coagulation reaction measurement in step 1, and when data necessary for calculating the coagulation time of the test sample has been obtained, the coagulation reaction measurement of that sample is terminated and the coagulation reaction measurement of the next sample is started.

[0022] In step 2a, measurement data from the coagulation reaction measurement is obtained. This data is, for example, data reflecting the coagulation reaction process of the sample obtained by the APTT measurement in step 2 described above. For example, data showing the change over time in the progress of the coagulation reaction (e.g., the amount of scattered light) after the addition of calcium chloride solution is obtained from a sample containing a test specimen and a coagulation time measurement reagent. The data obtained from these coagulation reaction measurements is also referred to as coagulation reaction information in this specification.

[0023] An example of the clotting reaction information acquired in step 2a is shown in Figure 3. Figure 3 is a clotting reaction curve based on the amount of scattered light, with the horizontal axis indicating the time elapsed after the addition of calcium chloride solution (clotting reaction time) and the vertical axis indicating the amount of scattered light. As time passes, the clotting reaction of the mixed solution progresses, so the amount of scattered light increases. In this specification, a curve showing the change in the amount of clotting reaction versus the clotting reaction time is referred to as a clotting reaction curve.

[0024] A coagulation reaction curve based on the amount of scattered light as shown in Fig. 3 is usually sigmoidal. On the other hand, a coagulation reaction curve based on the amount of transmitted light is usually inverted sigmoidal. In the rest of this specification, data analysis using a coagulation reaction curve based on the amount of scattered light as coagulation reaction information will be described.

[0025] If necessary, the coagulation reaction curve may be pre-processed (step 2b). The pre-processing may include smoothing to remove noise or zero-point adjustment. FIG. 4 shows an example of the coagulation reaction curve of FIG. 3 that has been pre-processed (smoothing and zero-point adjustment). Any of the known noise removal methods may be used for the smoothing. As shown in FIG. 3, the mixed liquid containing the test specimen originally scatters light, so that the amount of scattered light at the start of the measurement (time 0) is greater than 0. By the zero-point adjustment after the smoothing, the amount of scattered light at time 0 is adjusted to 0 as shown in FIG. 4. FIGS. 5A and 5B show partial enlarged views of the coagulation reaction curve of FIG. 3 before and after pre-processing, respectively. In FIG. 5B, the data of FIG. 5A has been smoothed and zero-point adjusted.

[0026] The height of the coagulation reaction curve depends on the fibrinogen concentration of the test specimen. Meanwhile, since there are individual differences in fibrinogen concentration, the height of the coagulation reaction curve differs depending on the test specimen. Therefore, in this method, if necessary, a correction process is performed in step 2c to convert the coagulation reaction curve after pretreatment into a relative value. This correction process makes it possible to obtain a coagulation reaction curve that is not dependent on the fibrinogen concentration, and thereby makes it possible to quantitatively compare the difference in the shape of the coagulation reaction curve after pretreatment between samples.

[0027] In one embodiment, the correction process corrects the coagulation reaction curve after pretreatment so that the maximum value becomes a predetermined value. Preferably, the correction process obtains a corrected coagulation reaction curve P(t) from the coagulation reaction curve after pretreatment according to the following formula (1). In formula (1), D(t) represents the coagulation reaction curve after pretreatment, Dmax and Dmin represent the maximum and minimum values ​​of D(t), respectively, Drange represents the range of change of D(t) (i.e., Dmax-Dmin), and A is an arbitrary value representing the maximum value of the corrected coagulation reaction curve. P(t)=[(D(t)-Dmin) / Drange]×A (1)

[0028] As an example, Fig. 6 shows data corrected so that the coagulation reaction curve shown in Fig. 4 has a maximum value of 100. Note that, although the corrected values ​​are corrected to be between 0 and 100 in Fig. 6, other values ​​(for example, between 0 and 10000, i.e., A = 10000 in formula (1)) may also be used. Also, this correction process does not necessarily have to be performed.

[0029] Alternatively, the above-mentioned correction process may be performed on the waveform related to the coagulation rate, which will be described later, or on parameters extracted from the waveform. For example, a waveform related to the coagulation rate can be calculated for a pre-processed coagulation reaction curve D(t) that is not subjected to correction process, and then converted into a value equivalent to P(t). Alternatively, a parameter can be extracted from the waveform related to the coagulation rate, and then the value of the parameter can be converted into a value equivalent to P(t).

[0030] In this specification, the above-mentioned corrected coagulation reaction curve and uncorrected coagulation reaction curve are also called corrected zero-order curve and uncorrected zero-order curve, respectively, and are also collectively called "zero-order curve". In addition, in this specification, the first-order differential curves of the corrected zero-order curve and the uncorrected zero-order curve are also called corrected linear curve and uncorrected linear curve, respectively, and are also collectively called "linear curve".

[0031] 2.2) Calculation of waveforms related to coagulation rate In step 2d, a waveform related to the coagulation rate is calculated. In this specification, the waveform related to the coagulation rate includes an uncorrected linear curve and a corrected linear curve. The uncorrected linear curve represents a value obtained by first differentiating the coagulation reaction curve (uncorrected zero-order curve), i.e., a rate of change (coagulation rate) of the amount of coagulation reaction at any coagulation reaction time. The corrected linear curve represents a value obtained by first differentiating the corrected coagulation reaction curve (corrected zero-order curve), i.e., a relative rate of change of the amount of coagulation reaction at any coagulation reaction time. Therefore, the waveform related to the coagulation rate can be a waveform representing the coagulation rate or its relative value in the coagulation reaction of the sample. In this specification, the value representing the progress of blood coagulation, including the coagulation rate and its relative value represented by the linear curve, may be collectively referred to as a first derivative value. The differentiation of the coagulation reaction curve or the corrected coagulation reaction curve (uncorrected and corrected zero-order curves) can be performed using a known method. FIG. 7 shows a corrected linear curve obtained by first differentiating the corrected zero-order curve shown in FIG. 6. The horizontal axis of FIG. 7 represents the coagulation reaction time, and the vertical axis represents the first derivative value.

[0032] 2.3) Parameter Extraction In step 2e, parameters characterizing the waveform related to the clotting rate are extracted. More specifically, in the parameter extraction step, a predetermined calculation target area is extracted from the waveform related to the clotting rate, and then a weighted average time for the calculation target area is calculated. Based on the weighted average time, the blood clotting time of the test sample can be determined (step 3). The parameters are described below.

[0033] First, a procedure for extracting a predetermined calculation target area from a waveform related to a coagulation rate will be described. The calculation target area refers to an area of ​​the waveform related to the coagulation rate where the waveform is equal to or greater than a predetermined lower limit. More specifically, the calculation target area is an area (segment) of F(t) that satisfies F(t)≧Vmax×x% when the waveform (linear curve) related to the coagulation rate is F(t) (t=time) and the maximum value of F(t) is Vmax. More specifically, the calculation target area is an area (segment) of a linear curve F(t) that satisfies Vmax≧F(t)≧Vmax×x%. Therefore, "Vmax×x%" represents the lower limit of the calculation target area. The calculation target area will be described with reference to FIG. 8. FIG. 8 shows a linear curve F(t) (t=time) and a maximum value Vmax of F(t). In addition, a base line indicating Vmax×x% is illustrated by a dotted line, and the time points t1 and t2 at which F(t)=Vmax×x% are shown. The calculation target region is the region where F(t) is greater than or equal to the baseline and less than or equal to Vmax (F(t)≧Vmax × x%, t1≦t≦t2).

[0034] The weighted average point (vT, vH) corresponds to the "weighted average value" of the calculation target area. The coagulation reaction time (t) at the weighted average point is the weighted average time vT. In other words, the weighted average time vT is the time from the start of the coagulation reaction to the weighted average point, and is the x-coordinate of the weighted average point. Also, vT is the center of gravity in the horizontal direction of the calculation target area. The weighted average height vH is the y-coordinate of the weighted average point.

[0035] In this specification, the calculation target area based on a predetermined lower limit value and vT and vH derived from the calculation target area may be expressed according to the percentage of the lower limit value Vmax. For example, a calculation target area with a lower limit value of x% of Vmax may be referred to as an "x% calculation target area", and vT and vH derived from the "x% calculation target area" may be referred to as vTx% and vHx%, respectively. For example, a calculation target area with a lower limit value of 20% of Vmax is referred to as a 20% calculation target area, and vT for the calculation target area is represented as vT20%. Figure 8 shows the weighted average score (vTx%, vHx%) for the calculation target area of ​​F(t) at the lower limit value x%.

[0036] The weighted average time vT and the weighted average height vH for the first-order curve can be obtained by the following procedure. First, let the maximum value of the first-order curve F(t) be Vmax, the lower limit value of the calculation target range be x% of Vmax, and the data group of time t that satisfies F(t) ≥ Vmax × x% be t[t1, …t2] (t1 < t2). That is, F(t1) = Vmax × x%, F(t2) = Vmax × x%, and the data group at times t1 to t2 is t[t1, …t2] (t1 < t2). At this time, the weighted average time vT and the weighted average height vH are calculated by the following equations (2) and (3), respectively. From the obtained vT and vH, the weighted average point (vTx%, vHx%) for the x% calculation target range is derived.

[0037]

Equation

[0038] The lower limit value of the calculation target range can be determined within the range greater than 0% and less than Vmax of Vmax. The calculation target range reflects the shape of the first-order curve. The larger the lower limit value, the more the upper shape of the first-order curve is reflected in the calculation target range. In the method of the present invention, the lower limit value ("Vmax × x%") of the calculation target range for calculating the weighted average time used for measuring the coagulation time is preferably a predetermined value set within the range of 5 to 95% of Vmax (that is, x = 5 to 95). More specifically, the lower limit value of the calculation target range for calculating the weighted average time used for measuring the coagulation time is preferably 5 to 95% of Vmax (x = 5 to 95), more preferably 5 to 50% of Vmax (x = 5 to 50), and even more preferably 10 to 35% of Vmax (x = 10 to 35) when the coagulation time is APTT, and is preferably a predetermined value set within the range of 5 to 95% of Vmax (x = 5 to 95), more preferably 10 to 80% of Vmax (x = 10 to 80), and even more preferably 25 to 50% of Vmax (x = 25 to 50) when the coagulation time is PT.

[0039] Figure 9 shows the relationship between the calculation target area of ​​the linear curve and the calculated weighted average point. In Figure 9, the upper, middle, and lower parts show the calculation target area of ​​the linear curve and the weighted average point (black circle) when the lower limit value is 5%, 40%, and 75% of Vmax, respectively. As the calculation target area changes, the position of the weighted average point changes as shown in Figure 9.

[0040] In the above-mentioned FIGS. 8 and 9, the calculation target area and its weighted average point have been described using a corrected linear curve as an example, but similar parameters can also be calculated using an uncorrected linear curve.

[0041] 3. Clotting Time Measurement As shown in the examples below, the weighted average time vT calculated by the above procedure has a high correlation with the blood clotting time of the test sample. Therefore, the clotting time of the test sample can be determined based on the weighted average time vT. For example, when the clotting time is APTT or PT, the weighted average time vT can be determined as the clotting time. In the method of the present invention, by calculating the weighted average of the linear curve of the clotting reaction, it is possible to perform a more reliable clotting time measurement that is less susceptible to the effects of measurement noise and bimodal peaks caused by clotting abnormalities, compared to the case of simply detecting the peak of the linear curve.

[0042] Furthermore, in the method of the present invention, it becomes possible to measure the clotting time when the linear curve of the clotting reaction reaches the maximum value Vmax and then falls below the lower limit of the calculation range (Vmax × x%). Therefore, in the method of the present invention, it is not necessary to continue measurement until the clotting reaction reaches a plateau, as in the conventional percentage method. Furthermore, even when multiple samples are analyzed using an automatic analyzer, the method of the present invention does not require setting a long measurement time in preparation for abnormal samples with low coagulation ability, as in the conventional percentage method. Therefore, according to the present invention, the sample analysis time can be shortened or optimized, improving the efficiency of analysis.

[0043] 4. Clotting factor concentration measurement Normal blood contains clotting factors such as clotting factors I to XIII, and abnormalities or deficiencies of these clotting factors result in abnormalities in clotting ability. Usually, the clotting factor concentration of a test specimen can be measured based on the clotting time of a measurement sample prepared from the test specimen using a dedicated reagent for each clotting factor. Therefore, the clotting factor concentration of the test specimen can be measured using the clotting time of the measurement sample measured based on the weighted average time by the method of the present invention. Usually, the clotting factor concentration is measured based on a calibration curve showing the relationship between the clotting time and the clotting factor concentration. Therefore, the clotting factor concentration of the test specimen can be measured by applying the clotting time of the measurement sample measured by the method of the present invention to the calibration curve created in advance. Preferred examples of clotting factors measured by the method of the present invention include factor I (fibrinogen), factor VIII, factor IX, etc.

[0044] In the method of the present invention, the lower limit of the calculation range for calculating the clotting time (weighted average time) used in measuring the coagulation factor concentration ("Vmax × x%) is a predetermined value set preferably within the range of 5 to 95% of Vmax (i.e., x = 5 to 95), more preferably within the range of 30 to 95% of Vmax (i.e., x = 30 to 95), and even more preferably within the range of 60 to 75% of Vmax (i.e., x = 60 to 75).

[0045] 5. Application to other coagulation reaction measurement methods The blood clotting time measurement method of the present invention has been described above by taking the case of clotting reaction measurement based on the amount of scattered light as an example. However, a person skilled in the art can apply the method of the present invention to a blood clotting time measurement method using other clotting reaction measurement methods (e.g., blood clotting reaction measurement methods based on transmittance, absorbance, viscosity, etc.). For example, the linear curve F(t) obtained from an inverse sigmoid clotting reaction curve based on the amount of transmitted light has a positive and negative opposite to that based on the amount of scattered light described above. In such a case, it is clear to a person skilled in the art that the sign of F(t) is reversed in the parameter calculation, for example, the maximum value Vmax is replaced by the minimum value Vmin, and the x% calculation target range is a region that satisfies F(t)≦Vmin × x%, etc. EXAMPLES

[0046] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples.

[0047] Example 1. Weighted average time based clotting time (APTT) measurement 1. Method 1.1) Sample A total of 24 samples were used as test samples, including 9 normal plasma samples and 15 abnormal plasma samples with prolonged APTT. Normal plasma was Normal Donor Plasma manufactured by CliniSys Associates, Ltd. Abnormal plasma was used, including 2 samples each of FVIII-deficient plasma, FIX-deficient plasma, FXI-deficient plasma, and FXII-deficient plasma as coagulation factor deficient plasma (Factor Deficient Plasma), 2 samples of lupus anticoagulant plasma, and 5 samples of unfractionated heparin-containing plasma (Anticoagulant Plasma) (all manufactured by CliniSys Associates, Ltd.).

[0048] 1.2) Reagents Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.) was used as the APTT reagent.

[0049] 1.3) Clotting reaction measurement The coagulation reaction was measured using an automatic blood coagulation analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the sample was dispensed into a cuvette (reaction vessel) and heated at 37°C for 45 seconds, then 50 μL of APTT reagent heated to about 37°C was added to the cuvette, and after 171 seconds, 50 μL of calcium chloride solution was added to start the coagulation reaction. The reaction was performed while maintaining the temperature at about 37°C. The coagulation reaction was measured (photometrically) by irradiating the cuvette with light from an LED light with a wavelength of 660 nm as a light source and measuring the amount of scattered light at 90 degrees side at 0.1 second intervals. The maximum measurement time was 360 seconds (3600 data points, 0.1 second intervals).

[0050] 1.4) APTT measurement (percentage method) The APTT of each specimen was measured by the percentage method. That is, the time point when the scattered light amount reached the maximum value within the measurement time was determined as the end point of the coagulation reaction, and the time point when the scattered light amount reached 50% of the scattered light amount at the end point of the coagulation reaction was determined as the APTT. Table 1 shows the types and numbers of the test specimens, and the minimum and maximum values of APTT in the specimens of each type.

[0051]

Table 1

[0052] 1.5) Creation of coagulation reaction curve After performing smoothing processing including noise removal on the photometric data from each specimen, zero-point adjustment processing was performed so that the scattered light amount at the start point of photometry became 0, and the coagulation reaction curve P(t) was calculated. Subsequently, correction was performed so that the maximum value Pmax of the coagulation reaction curve became 100, and the obtained corrected coagulation reaction curve (corrected zero-order curve) was differentiated by the first order to calculate the corrected first-order curve.

[0053] 1.6) Calculation of weighted average time vT From the first-order curves obtained from each specimen, the weighted average times (vT5% - vT95%) for the 5 - 95% calculation target range were calculated. The lower limit value x% of the calculation target range was set at 19 levels of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% of the maximum height Vmax (100%) of the first-order curve F(t), and the times t1 and t2 (t1 < t2) that satisfy F(t) = Vmax × x% were obtained. Using the above formula (2), 19 weighted average times vT5% - vT95% were calculated from each of the 24 specimens.

[0054] 2. APTT measurement based on weighted average time 2.1) Correlation between weighted average time and APTT The correlation between the APTT measured by the percentage method and the weighted average time was evaluated. A linear regression analysis was performed with the APTT measured by the percentage method for each of the weighted average times for the 5% to 95% calculation range (vT5% to vT95%), and the slope, intercept, and correlation coefficient of the regression line were calculated.

[0055] FIG. 10 shows the linear regression line of vT20% in the 20% calculation range against the APTT by the percentage method for 24 samples. vT20% had a high correlation with the APTT by the percentage method. FIG. 11 shows the slope, intercept, and correlation coefficient of the linear regression line of vT (vT5% to vT95%) in the 5 to 95% calculation range against the APTT by the percentage method. Between the 5 to 95% calculation range, the slope of the regression line was 0.95 to 1.01, the intercept was -2.0 to 0.4, and the correlation coefficient was 0.998 to 1.000 (FIGS. 11A to 11C). It was confirmed that in all conditions from 5% to 95% of the calculation range, the APTT measurement method based on the weighted average time has the performance that meets the Ministry of Health, Labor and Welfare's in vitro diagnostic drug approval standard "a correlation coefficient of 0.9 or more and a regression line slope of 0.9 to 1.1 compared to the control measurement method." These results demonstrated that APTT can be measured based on vT.

[0056] 2.2) Measurement accuracy Weighted mean times for 24 samples in the 5-95% calculation range were compared with the control (APTT by percentage method). Each row in the table in Figure 12AB represents the weighted mean time for each sample (vT5%-vT95%), and is shown in gray if the difference between the weighted mean time and the control was within ±5% (Figure 12A) and ±2.5% (Figure 12B) of the control. In the 5-50% calculation range, the weighted mean times for all samples matched the control within ±5% error, and in the 10-35% calculation range, the weighted mean times for all samples matched the control within ±2.5% error.

[0057] Example 2 Clotting Time (PT) Measurement Based on Weighted Mean Time 1. Method 1.1) Sample A total of 23 test samples were used: 9 normal plasma samples and 14 abnormal plasma samples with prolonged PT. Plasma from healthy individuals was used as normal plasma. Plasma from patients administered the anticoagulant warfarin was used as abnormal plasma. Five samples with PT-INR values ​​indicating blood warfarin concentrations of 1 to 2, five with PT-INR values ​​of 2 to 3, and four with PT-INR values ​​of 3 to 4 were used.

[0058] 1.2) Clotting reaction measurement The coagulation reaction was measured using an automatic blood coagulation analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the sample was dispensed into a cuvette (reaction vessel) and heated at 37°C for 45 seconds, and then 100 μL of thromboplastin solution heated to approximately 37°C was added to the cuvette to start the coagulation reaction. The reaction was carried out while maintaining the temperature at approximately 37°C. The coagulation reaction was measured (photometrically) by irradiating the cuvette with light from an LED light with a wavelength of 660 nm as a light source and measuring the amount of scattered light at 90 degrees side at 0.1 second intervals. The maximum measurement time was 300 seconds (3000 data points, 0.1 second intervals).

[0059] 1.3) PT measurement (percentage method) The PT of each sample was measured by the percentage method. The point at which the amount of scattered light reached its maximum value within the measurement time was determined as the end point of the coagulation reaction, and the point at which the amount of scattered light reached 45% of the end point of the coagulation reaction was determined as the PT. The types and numbers of samples tested, as well as the minimum and maximum PT values ​​for each type of sample, are shown in Table 2.

[0060] [Table 2]

[0061] 1.4) Calculation of weighted average time vT In the same manner as in 1.5) and 1.6) of Example 1, a corrected linear curve was calculated for the 23 samples, and the weighted average times vT5% to vT95% were calculated.

[0062] 2. PT measurement based on weighted average time 2.1) Correlation between weighted average time and PT Using the same procedure as in 2.1) of Example 1, a linear regression analysis was performed with respect to each of the weighted average times (vT5% to vT95%) for the 5% to 95% calculation range and the PT measured by the percentage method, and the slope, intercept, and correlation coefficient of the regression line were calculated.

[0063] FIG. 13 shows the linear regression line of vT30% in the 30% calculation range against the PT by the percentage method for 23 samples. vT30% had a high correlation with the PT by the percentage method. FIG. 14 shows the slope, intercept, and correlation coefficient of the linear regression line of vT (vT5%-vT95%) in the 5-95% calculation range against the PT by the percentage method. Between the 5-95% calculation range, the slope of the regression line was 0.94-1.05, the intercept was -0.1-0.7, and the correlation coefficient was all 1.000 (FIGS. 14A-C). It was confirmed that the PT measurement method based on the weighted average time has the performance that meets the Ministry of Health, Labor and Welfare's in vitro diagnostic drug approval standard "correlation coefficient 0.9 or more and regression line slope 0.9-1.1 compared to the control measurement method". These results showed that PT can be measured based on vT.

[0064] 2.2) Measurement accuracy Weighted mean times for 23 samples in the 5-95% calculation range were compared with the control (PT by percentage method). Each row in the table in Figure 15AB represents the weighted mean time for each sample (vT5%-vT95%), and is shown in gray if the difference between the weighted mean time and the control was within ±5% (Figure 15A) and ±2.5% (Figure 15B) of the control. In the 10-80% calculation range, the weighted mean times for all samples matched the control within ±5% error, and in the 25-50% calculation range, the weighted mean times for all samples matched the control within ±2.5% error.

[0065] Example 3 Fibrinogen concentration measurement based on weighted average time 1. Method 1.1) Sample A sample (sample 10) with a fibrinogen concentration ([Fbg]) of 980 mg / dL was prepared by adding human fibrinogen (Human Fibrinogen, Enzyme Research Laboratories, product name: FIB 2) to human fibrinogen-depleted plasma (Fibrinogen Deficient Human Plasma, Affinity Biologicals Inc., product name: Fg Deficient Plasma). As standard samples for creating a calibration curve, sample 10 was mixed with saline in volume ratios of 1:9, 7:3, and 10:0 to prepare three samples with fibrinogen concentrations ([Fbg]) of 98 mg / dL, 686 mg / dL, and 980 mg / dL, respectively. In addition, sample 10 was mixed with the human fibrinogen-depleted plasma in volume ratios ranging from 1:9 to 10:0 to prepare 10 concentration series samples with gradually different fibrinogen concentrations ([Fbg]) (Table 3).

[0066] [Table 3]

[0067] 1.2) Clotting reaction measurement The fibrinogen measurement reagent used was the thrombin reagent and specimen diluent attached to Coagpia Fbg (manufactured by Sekisui Medical Co., Ltd.). The coagulation reaction was measured using an automatic blood coagulation analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). 10 μL of the specimen and 90 μL of the specimen diluent were dispensed into a cuvette and heated at 37°C for 45 seconds, after which 50 μL of thrombin reagent heated to about 37°C was added to the cuvette to start the coagulation reaction. The reaction was carried out while maintaining the temperature at about 37°C. The coagulation reaction was measured by irradiating the cuvette with light from an LED light with a wavelength of 660 nm as a light source and measuring the amount of scattered light at 90 degrees side at 0.1 second intervals. The maximum measurement time was 300 seconds (3000 data points, 0.1 second intervals). The coagulation reaction was measured twice for each of the three standard specimens and the 10 concentration series specimens.

[0068] 1.3) Calculation of fibrinogen concentration by clotting time measurement (percentage method) The clotting times of three standard specimens and ten concentration series specimens were measured by the percentage method. That is, the time when the scattered light amount reached 63% of the end point of the clotting reaction was determined as the clotting time. The clotting time of each specimen was measured twice based on two clotting reaction measurements. The average value of the two measurements was calculated for the clotting times measured from the three standard specimens, and the logarithm of the average value was plotted against the logarithm of [Fbg] (mg / dL) of the standard specimen to create a calibration curve by the percentage method. The fibrinogen concentration of each concentration series specimen (calculated value of [Fbg] by the percentage method, mg / dL) was calculated according to the created calibration curve.

[0069] 1.4) Calculation of fibrinogen concentration by weighted mean time vT In the same manner as in 1.5) and 1.6) of Example 1, the weighted average times vT5% to vT95% were calculated twice for each of the 10 concentration series samples, and data on the weighted average times for the 20 samples was obtained. The weighted average times vT5% to vT95% were also calculated twice for each of the three standard samples, and the average value of the two measurements was calculated. The logarithm of the average value was plotted against the logarithm of [Fbg] (mg / dL) of the standard sample to create a calibration curve based on the weighted average time. The fibrinogen concentration of each concentration series sample (calculated value of [Fbg] based on the weighted average time, mg / dL) was calculated according to the created calibration curve.

[0070] 2. Evaluation of fibrinogen concentration measurements based on weighted average time 2.1) Correlation analysis FIG. 16 shows the linear regression line of the calculated [Fbg] value (mg / dL) by the weighted average time calculated from vT35% in the 35% calculation range against the calculated [Fbg] value by the percentage method based on the data of the concentration series specimens (n=10×2). The calculated [Fbg] value based on vT35% had a high correlation with the calculated [Fbg] value by the percentage method. FIG. 17 shows the slope, intercept, and correlation coefficient of the linear regression line of the calculated [Fbg] value by the percentage method against the calculated [Fbg] value calculated from vT (vT5%-vT95%) in the 5-95% calculation range. Between the 5-95% calculation range, the slope of the regression line was 0.94-1.02, the intercept was -31.5-51.0, and the correlation coefficient was 0.990-0.996 (FIGS. 17A-C). It was confirmed that under all conditions, from 5% to 95% of the calculation range, the fibrinogen concentration measurement method based on the weighted average time gives results equivalent to the standard method based on the percentage method, and therefore has performance that meets the Ministry of Health, Labor and Welfare's in vitro diagnostic drug approval standards of "a correlation coefficient of 0.9 or more and a regression line slope of 0.9 to 1.1 compared to the control measurement method." These results demonstrated that fibrinogen concentration can be measured based on vT.

[0071] 2.2) Measurement accuracy The calculated [Fbg] values ​​based on the weighted average time in the 5-95% calculation range for the concentration series sample data (10 samples × 2 in Table 3) were compared with the expected values ​​(Fbg concentrations of the samples shown in Table 3). Each row in the table in Figure 18AB represents the weighted average time (vT5%-vT95%) for each sample, and when the error between the calculated [Fbg] value based on the weighted average time and the expected value was within ±10% (Figure 18A) or ±5% (Figure 18B), it is shown in gray. In the 30-95% calculation range, the error of the calculated [Fbg] values ​​for all samples was within ±10%, and in the 60-75% calculation range, the error of the calculated [Fbg] values ​​for all samples was within ±5%.

Claims

1. A method for measuring blood coagulation time, comprising: measuring a coagulation reaction for a sample in which a test specimen and a coagulation time measurement reagent are mixed; calculating a weighted average time for an arithmetic operation target region of a waveform related to a coagulation rate from the obtained measurement data; and determining the blood coagulation time of the test specimen based on the weighted average time, wherein the arithmetic operation target region is a region in the waveform related to the coagulation rate where the waveform is equal to or greater than a predetermined lower limit value. Method.

2. The method according to claim 1, wherein the waveform related to the coagulation rate is a coagulation reaction curve or a first derivative curve of a relative value thereof.

3. When the weighted average time is such that the waveform related to the coagulation rate is F(t) (t is time), and F(t) is x% of the maximum value (x is a predetermined value set in the range of 5 to 95), and the times are t1 and t2 (t1 < t2), the following formula: [0010] The method according to claim 1 or 2, which is represented by

4. The method according to any one of claims 1 to 3, wherein the test specimen is plasma.

5. The method according to any one of claims 1 to 4, wherein the blood coagulation time is an activated partial thromboplastin time (APTT), a prothrombin time (PT), or a coagulation time in fibrinogen concentration measurement.

6. A method for measuring a coagulation factor concentration, comprising measuring the coagulation factor concentration of a test specimen based on the blood coagulation time of the test specimen measured by the method according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the test specimen is plasma.

8. The method according to claim 6 or 7, wherein the coagulation factor is fibrinogen.

9. The method according to claim 8, wherein the blood coagulation time is a coagulation time in fibrinogen concentration measurement.

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